Orally Administered Bisphenol A Impairs Normal Thyroid Functions Altering Thyroid Hormones Homeostasis in Female Wistar Rats

Received 16 Feb, 2021

Accepted 12 Apr, 2021

Published 15 Jun, 2021

ABSTRACT
Background Objective: Bisphenol A (BPA) is an endocrine-disrupting chemical used on a wide range in the industry. Several studies reported that BPA may cause cardiovascular disorders in humans and animals. The present study aims to investigate the effect of BPA on the thyroid gland of female rats. Materials and Methods: The rats received a daily oral administration of BPA (0.05 – 1.0 mg kg^-1 for 13 weeks). Thyroid Stimulating Hormone (TSH), Thyroxine (T₄) and Triiodothyronine (T₃) were assayed using an Auto chemical analyzer and data obtained were subjected to statistical analysis with the Statistical Package for the Social Sciences (SPSS) software. Results: It was found that BPA at the eleven studied doses induced a significant increase in the Thyroxin Stimulating Hormone (TSH), Free Thyroxin (FT₄), Total Thyroxin (TT₄) level, while the bound thyroxin is low compared to the control. The Free Triiodothyronine (FT₃) and Total Triiodothyronine (TT₃) were initially low at group 1 but at the other doses, they were on the increase. The bound triiodothyronine is lower when compared to that of the control throughout the studied time intervals. Conclusion: These results suggest that BPA has thyroid toxic effects which are mediated by the oxidative stress resulting from the overproduction of free radicals, BPA may also participate in the thyroid gland function disturbances.

INTRODUCTION

Bisphenol-A is an organic compound that has two phenol functional groups. It is prepared by the condensation reaction of acetone, and two equivalents of phenol¹ with hydrochloric acid² (Fig. 1). Bisphenol-A has a phenolic odour witha melting point of 155°C and a specific gravity of 1.060-1.195 gcm^-13,4. It is soluble in nonpolar solvent⁵. Aerobic biodegradation and biodegradation half-life for BPA in river water and soil is about 4.5 days³ and the photo-oxidation half-life for BPA in the air is about 4 hrs⁶. It is known that the global population is subject to repeated exposure to BPA is via the diet⁷ with detectable metabolites in the urine⁸. Daily dietary intake of about 0.2 μg kg^-1 body weight in breastfed babies and 1.5 μg kg^-1 body weight in adults⁹ relevant doses of BPA causes increases in weight and size of the prostate gland, decreases in sperm efficiency, earlier puberty¹⁰, and abnormalities in the oocytes¹¹. Invernizzi¹² showed that BPA triggers ductal and alveolar structures proliferations, development of ductal hyperplasia¹³, modifications of the mammary gland architecture¹⁴, mammary carcinogenesis¹⁵, inflammatory cytokine dysregulation¹⁶, and mitochondrial-mediated apoptosis¹⁷. Health implications associated with BPA exposure include diabetes¹⁸, cardiovascular disease¹⁹, altered liver enzymes activities²⁰ and obesity-promoting effects²¹. BPA alters glucose homeostasis, increased pancreatic insulin and induced insulin resistance²², BPA induces oxidative stress²³, coronary artery disease²⁴, activates Maxi-K ion channels²⁵, increased BP and decreased Heart Rate (HR)²⁶, increased risk of hypertension²⁷, decreased efficiency of sperm production²³ and increased ovarian cancer cell proliferation²⁸. According to this, Miyagawa et al.²⁹ reported impaired memory, increased aggressiveness³⁰, alters anxiety³¹, loss or reduction of sexual dimorphisms³², loss of sex difference in corticotrophin-releasing hormones³³, reduced the amount of tyrosine by dioxylase-immunoreactive³⁴. This study aims to establish the possible effects and physiological disposition of Bisphenol-A on sex and thyroid hormones, in female Wistar albino rats.

(CH₃)₂CO+2C₆H₅OH (CH₃)₂C(C₆H₄OH)₂+H₂O

Fig. 1: Structure of Bisphenol-A

MATERIALS AND METHODS

Study area: The study was carried out at Applied Biochemistry Lab, Nnamdi Azikiwe University, Awka, Nigeria and Biochemistry Lab, Gregory University Uturu, AbiaState, Nigeria from June-September, 2017.

Methodology: Total 60 non-pregnant female rats of 5 weeks age were acclimatized in the laboratory for 7 days and randomly divided into 11 experimental groups of 5 rats each and respectively administered; 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mg of BPA kg^-1 b.wt.day^-1. The first group which served as control did not receive any treatment, but distilled water instead. The graded doses of BPA were dissolved in distilled water and administered by oral gavage using an intubation cannula (Lars Medicare Pvt. Ltd, New Delhi, India). Blood was obtained from the tail of the various groups by capillary action, weekly, after BPA administration for 13 weeks. Blood samples were processed for clinical assay.

Animals have housed in aluminium wire-mesh cages in a well-ventilated animal house with a 12 hrs dark/light cycle and at room temperature and were provided commercial rat pellets (Vital feed from Vital group of Company, Nigeria) and water ad libitum.

At the end of the experiments serum TSH, Thyroxine (T₄) and Triiodothyronine (T₃) were assayed using an Auto chemical analyzer (Lx20 pro-Autoanalyzer, Beckman Coulter, Woerden, Netherland and Chemwell chemical Analyzer, Manufacturer: Roche Hitachi, GMI.). All reagents were commercially obtained as already prepared kits. The kits for TSH, Thyroxine (T₄) and Triiodothyronine (T₃) were purchased from Phoenix Pharmaceuticals, Burlingame, CA; Enzo Life Sciences Inc, Boulevard Farmingdale, NY; Diagnostic automation/Cortez Diagnostics Inc, Calabasas, CA. Individual tests were carried out according to the kit specifications.

Statistical analysis: Differences between obtained values (mean±SD) were carried out by one-way analysis of variance (ANOVA) using SPSS software version 20.0 followed by the Tukey-Kramer multiple comparison test. At p≤0.05 was taken as a criterion for a statistically significant difference.

RESULTS

Thyroid-stimulating hormone: The experimental groups that were administered with 0.2–1 mg kg^-1 BPA showeda dose-dependent significant increase in the concentration of thyroid-stimulating hormones when compared with the control (Fig. 2), the increase over time appeared to be relatively constant except for 0.6 mg kg^-1 BPA test group that showed a constant increase in TSH with time and 1 mg kg^-1 BPA test group that showed high levels of the thyroid-stimulating hormone at week 13. The test exposed 0.05 mg kg^-1 BPA showed a nonsignificant increase in TSH that remained relatively constant with time. The group that received 0.1 mg kg^-1 BPA responded differently during the study, it was observed that at the first week of exposure there was an increase in TSH, which gradually decreased through week 3, but surprisingly a consistent rise in the concentration of TSH recorded from week 4-13. All the weeks of exposure showed a characteristic dose-dependent effect of BPA on TSH, as the administered dose increases, the effect of BPA on TSH increases. The weeks of exposure showed sensitivity at two-point (0.1 and 0.6) but was constant at other points of exposure.

Fig. 2: Graphical representation of thyroid-stimulating hormone level

Total Thyroxine (TT₄): All the experimental groups that were administered with (0.05–1 mg kg^-1) BPA showed a significant increase in the concentration of TT₄ when compared with the control (Fig. 3), amidst the increase TT₄, the test groups exposed to 0.05-0.4 and 0.6-1 mg kg^-1 showed a dose-dependent effect, while the TT₄ level of the test groups exposed to 0.5 mg kg^-1 BPA decreased relative to those of 0.4 mg kg^-1 again those of 0.6 mg kg^-1 test group decreased further relative to those of 0.5 mg kg^-1 BPA test group. All the weeks of exposure showed a characteristic high but not dose-dependent effect of BPA on TT₄. The weeks of exposure showed a non-sensitivity response with the lest observed effect at point 0.9.

Fig. 3: Graphical representation of Total Thyroxine (TT4) level

Free Thyroxine (FT₄): All the experimental groups that were administered with (0.05–1 mg kg^-1) BPA showed a significant increase in the concentration of FT₄ when compared with the control (Fig. 4), which appears to be relatively constant with the time of exposure. Amidst the increase FT₄, the test groups exposed to 0.05-0.4, and 0.6-1 mg kg^-1 showed a dose-dependent effect, while the FT₄ level of the test groups exposed to 0.5 mg kg^-1BPA decreased relative to those of 0.4 mg kg^-1 again those of 0.6 mg kg^-1 test group decreased further relative to those of 0.5 mg kg^-1 BPA test group. All the weeks of exposure showed a characteristic high and dose-dependent effect of BPA on FT₄ on the 0.05–0.4 doses of BPA. The weeks of exposure showed a decrease response at point 0.5 -0.6 mg kg^-1 BPA and tend to rise from 0.7 1 mg kg^-1.

Fig. 4: Graphical representation of Free Thyroxine FT4 level

Conjugated Thyroxine (BT₄): All the experimental groups that were administered with (0.05–1 mg kg^-1) BPA showed a significant noncharacteristic decrease in the concentration of conjugated thyroxine (BT₄) when compared with the control (Fig. 5). All the weeks of exposure showed a noncharacteristic low but not the dose-dependent effect of BPA on BT₄. The weeks of exposure showeda slight sensitivity response with the highest observed effect at point 1 mg kg^-1.

Fig. 5: Graphical representation of conjugated thyroxine

Total Triiodothyronine (TT₃): The experimental groups that were administered with 0.1–1 mg kg^-1 BPA showed a dose-dependent significant increase in the concentration of Total Triiodothyronine (TT₃) when compared with the control (Fig. 6), the increase over time appeared to be relatively constant except 0.7 and 0.9 mg kg^-1 BPA test group that showed a decrease in TT₃ relative to 0.6 and 0.8 mg kg^-1 BPA test group respectively. The test exposed 0.05 mg kg^-1 BPA showed a nonsignificant decrease in TT₃ that remained relatively constant with time (Fig. 6). All the weeks of exposure showed a characteristic relative dose-dependent effect of BPA on TT₃, as the administered dose increases, the effect of BPA on TT₃ increases. The weeks of exposure showed a low level of TT₃ at 0.7 points, falling below the control at point 0.05.

Fig. 6: Graphical representation of total Triiodothyronine (TT3) level

Free Triiodothyronine (FT₃): The experimental groups that were administered with 0.1–1mg kg^-1BPA showed a non-dose-dependent significant increase in the concentration of Free Triiodothyronine (FT₃) when compared with the control (Fig. 7). The test exposed 0.05 mg kg^-1 BPA showed a nonsignificant decrease in FT₃ that remained relatively constant with time (Fig. 7). All the weeks of exposure showed a characteristic relative effect of BPA on FT₃. The weeks of exposure showed a low level of FT₃ at 0.7 points, falling below the control at point 0.05.

Fig. 7: Graphical representation of Free Triiodothyronine (FT3) level

Conjugated Triiodothyronine (BT₃): The experimental groups that were administered with 0.1–1 mg kg^-1 BPA showed a significant decrease in the concentration of Conjugated Triiodothyronine (BT₃) when compared with the control (Fig. 8). All the weeks of exposure showed a characteristic relative non-dose-dependent effect of BPA on BT₃.

Fig. 8: Graphical representation of Conjugated Triiodothyronine (BT3) level

DISCUSSION

In this research, it was observed that the Thyroxin Stimulating Hormone (TSH), Free Thyroxin (FT₄), Total Thyroxin (TT₄) level were higher than that of the control, while the bound thyroxin is low compared to the control. The Free Triiodothyronine (FT₃) and Total Triiodothyronine(TT₃) were initially low at group 1 but at the other doses, they were on the increase. The bound triiodothyronine is lower when compared to that of the control. There is increasing evidence that exposure to BPA, can impair normal thyroid function. From this experiment, BPA alters bound circulating and thyroid hormone concentrations and BPA were observed to alter thyroid hormone status.

Previous studies have revealed that BPA exposure altered thyroid hormones by increasing Free Triiodothyronine concentrations (FT₃)³⁵, increased thyroid function³⁵, altered total T₄ and TSH³⁶, altered FT₄ level, and TSH³⁷, reduced bound T₄³⁸. Almudena³⁹ showed an increase in free T₃ levels. Du et al.⁴⁰ revealed a decrease in serum bound T₄ level. Zoeller et al.⁴¹, reported plasma free T₄ increase.

BPA elicits its effect on the T₄ hormone of the thyroid system as proposed: BPA inhibits the activity of the Thyroid Peroxidase (TPO) enzyme of the thyroid follicles, a key enzyme involved in the synthesis of T₃ and T₄⁴². BPA affects the thyroid receptor β physiology (TRβ), by repressing the transcription of TRβ and having an antagonistic effect on the TRβ, thereby interfering with the negative feedback that the thyroid hormones have on TSH release. Again, BPA alteration of thyroid hormones could be due to its ability to bind competitively with thyroid hormone transport proteins and induce UDP-glucuronosyltransferase activity which amplified biliary excretion of thyroxine⁴³. Also, BPA causes direct damage to the thyroid gland⁴⁰. Increase estrogen triggers decreases serum bound T₄, as proposed by Zhai et al.⁴⁴.

For Triiodothyronine (T₃) the effect of exposure to BPA could be explained by an increase of free T₃ synthesis in the thyroid follicles and higher T₄ deiodination in the liver. BPA up-regulate genes involved in the synthesis of thyroid hormones in the thyroid follicle⁴⁵, which gives support that BPA could trigger an increase of T₃ synthesis. Also, considering the similar structure of BPA and T₃, BPA could impair thyroid hormone action by inhibiting T₃ binding to the TR and by suppressing its transcriptional activity. The BPA may silence or inhibit the expression of some genes that govern normal thyroid development.

BPA have a direct effect on thyroid follicular cell and leads to an altered expression of the genes involved in thyroid hormones synthesis. Other potential mechanisms for the effect of BPA on thyroid hormones include inhibiting thyroid hormone pathways⁴⁶ and thyroid hormone receptor (TR) transcription suppression⁴², competitive binding with thyroid hormone for the thyroid plasma transporter⁴⁷. Also, BPA act as an antagonist of the thyroid hormone receptor because of its structural similarity to thyroid hormone. BPA inhibits thyroperoxidase activity, and accordingly block thyroid-induced metamorphism⁴⁸. At the receptor level, BPA bind to the thyroid hormone receptor as a ligand and act as an antagonist, Inhibiting TR-mediated transcriptional activity^49,50. BPA displace TH from serum binding proteins and because it can displace TH from these binding proteins, it causes a decline in the bound serum hormone levels^5,40. Zhai et al.⁴⁴ suggested another explanation to these results, which is the enhanced T₄ glucuronidation in the liver and excretion of T₄ into the bile, decreasing the amount of bound T₄ in plasma. Another explanation could be an increase of T₄ synthesis in the thyroid gland, driven by higher TSH levels induced after BPA exposure as observed in the study.

BPA have effects on the thyroid receptor β (TRβ), by repressing the transcription of the thyroid receptor β⁴² and having an antagonistic role on the thyroid receptor β. There is an interference on the negative feedback that the thyroid hormones carry out on TSH release⁴¹, accelerated embryonic development and advanced hatching⁵¹, and interfered with T₃ action during metamorphosis processes⁵². There is increasing evidence that exposure to BPA, impair normal thyroid function⁵³, reduced bound circulating and tissue thyroid hormone concentrations^54,55. Bisphenol-A has shown a high affinity for TTR and prealbumin, competing with thyroid hormones for these plasma transporters and decreasing plasma bound thyroid levels^5,56. BPA alter thyroid hormone status⁵⁷, increased thyroid function³⁵. An inverse relationship between urinary BPA and total T₄ and TSH have been reported³⁶. There is a significantly negative correlation between serum BPA and FT₄ level³⁷. BPA exposure up-regulates genes involved in the synthesis of thyroid hormones in the thyroid follicle⁴⁵, reduced bound T₄ and decreased TSH³⁸, decrease in serum bound T₄ level⁴⁰ and increase in free T₄ levels⁴¹. Studies have shown that BPA can compete with T₃ to bind with the thyroid plasma transporter, which could lead to a decrease in plasma bound T₃⁴⁷. BPA impair thyroid hormone action by inhibiting T₃ binding to the TR and by suppressing its transcriptional activity. Heimeier et al.⁴⁶, showed that doses of BPA affected the gene expression that is controlled by the T₃ hormone. BPA altered the expression of many genes known to be turned on by thyroid hormone. BPA has the potential to affect genes that are regulated by the thyroid hormone during human development. BPA also altered T₃ gene expression. Also, according to Heimeier et al.⁴⁶, BPA represents a serious risk to human development through the disruption of T₃ signaling pathways.

CONCLUSION

In conclusion, BPA is a potent inhibitor of the thyroid hormone, which directs development. BPA alters a subset of important genes controlled by T₃ that contribute to proper development, represents a serious risk to human development through disruption of TH signaling pathways. The study confirms past research showing BPA interferes with thyroid hormone activity. This research provides additional evidence into the fact that BPA interferes primarily with thyroid hormone level in addition to estrogen signals.

SIGNIFICANCE STATEMENT

BPA perturb thyroid hormone action throughout the body and interferes with thyroid hormone functions and homeostasis by inhibiting hormone synthesis, altering serum transport proteins, or increasing the catabolism of thyroid hormones. Suggestively, the altered thyroid functions can lead to thyroid abnormalities, the risk for developing thyroid nodules and obesity. This study will help the researcher to uncover the critical areas of Bisphenol-A toxicity on thyroid activity and thyroid hormone receptor that many researchers were not able to explore. Thus, a new theory on the association between BPA exposure and thyroid function may be arrived at.

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